Specificity of the protein kinase activity associated with the hemin-controlled repressor of rabbit reticulocyte.

Abstract

Highly purified preparations of hemin-controlled repressor of rabbit reticulocyte contain a 3':5'-cyclic AMP-independent protein kinase activity that phos horylates the low-molecular-weight (about 38,000) Globin synthesis in both rabbit reticulocytes (1, 2) and their cell-free lysates (3, 4) is controlled by the availability of hemin. The cessation of protein synthesis observed in the absence of added hemin is due to a block in initiation of new globin chains (3-7) and appears to involve the depletion of 40S ribosomal subunit-Met-tRNAf initiation complexes (8,9). An inhibitory protein, the hemin-controlled repressor (HCR), has been isolated from reticulocyte lysates incubated without hemin (10) which, when added to hemin-supplemented lysates, produced inhibition kinetics similar to those induced by hemin deficiency (11) and promotes the disappearance of 40S-Met-tRNAf initiation complexes (8,12). Hunt and coworkers (13,14) have reported that millimolar amounts of 3':5'-cyclic AMP (cAMP), 2-aminopurine, or GTP prevent the inhibition of protein synthesis observed in reticulocyte lysates incubated in the absence of hemin. They also observed that ATP counteracted the effect of GTP (14) In this communication we report that highly purified HCR preparations, while devoid of histone kinase activity, contain a cAMP-independent protein kinase activity that phosphorylates the 38,000-dalton polypeptide chain of IF-E2 and certain proteins associated with reticulocyte 408 ribosomal subunits that have been washed with 0.5 M KC1. Furthermore, goat IgG antibody against HCR neutralizes the above-mentioned kinase activity as well as the inhibitory activity of HCR while not having an effect on the histone kinase activity. METHODSFractionation of Reticulocyte and Artemia safina Components. Preparation of rabbit reticulocyte ribosomes has been described (18). A. salina ribosomes and their subunits were prepared by the procedure of Zasloff and Ochoa (19) as modified by Kramer et al. (20). Subunits of reticulocyte ribosomes were prepared by the procedure of Falvey and Staehelin (21) as modified by Obrig et al. (22). Ribosomal subunits, concentrated by centrifugation, were active in protein synthesis.The hemin-controlled repressor, HCR, from rabbit reticulocytes was purified essentially by the procedure of Gross and Rabinovitz (10) except that the order of the steps involved was altered. The postribosomal supernatant fraction of a 1:1 reticulocyte/water lysate was made 5 mM in N-ethylmaleimide, incubated for 15 min at 370, then made 5 mM in dithioerythritol and incubated at 370 for 10 min. The treated supernatant was then precipitated with ammonium sulfate between the levels of 0 and 50% saturation; the precipitate was fractionated by successive chromatography on DEAE-cellulose, hydroxylapatite, phosphocellulose, DEAE-cellulose, and Sepharose 6B. The resulting HCR preparation gives 50% inhibition of protein synthesis in a reticulocyte lysate at a concentration of 0.4 gg/ml. The 0.5 M KC1 ribosomal wash fraction of rabbit reticu...

ACCUMULATION OF MET-tRNA-80S RIBOSOME COMPLEX IN A CELL-FREE SYSTEM OF GLOBIN SYNTHESIS

“…These findings, however, cannot be considered to be caused by the action of hemin-controlled repressor (Maxwell et al, 1971;Adamson et al, 1972;Gross and Rabinovitz, 1972) since the inhibition of globin synthesis under hemin-deficient conditions was shown to result from impaired binding of the initiator tRNA to 40S ribosomal subunits by hemin-controlled repressor (Legon et al, 1973;Balkow et al, 1973). By use of a highly purified preparation of the hemin-controlled repressor, more recently, it was further shown to be a 3':5'-cyclic AMP-independent protein kinase phosphorylating the small subunit of the initiation factor that forms a ternary complex with Met-tRNA1 and GTP (Kramer et al, 1976;Levin et al, 1976;Raunu and London, 1976). To clarify the mechanism whereby the ternary complex reported herein accumulates, further studies are required on the regulation of the protein synthesis in a cellfree system employing subcomponents from the ribosomal wash fraction.…”

Reduced Formation of Initiation Complexes between Met-tRNAf and 40-S Ribosomal Subunits in Rabbit Reticulocyte Lysates Incubated at Elevated Temperatures. Activity of the Met-tRNAf Binding Factor

“…Moreover, addition of optimal amounts of control initiation factors, contained in the 0.5 M KCl ribosomal wash of unincubated lysates, to the 45 "C preheated lysates [2,6] or of a mixture of partially purified factors, including the Met-tRNAf binding factor, to preheated KCIwashed ribosomes and reticulocyte supernatant [7], fail to restore their rate of protein synthesis to the control level when subsequently assayed at 30-37 T. Incubation in the absence of haemin on the other hand results in the formation of a soluble inhibitor [4,8], identified as a protein kinase [9,10], which phosphorylates the small subunit of the initiation factor eIF-2; this inhibition can be overcome by the addition of large amounts of initiation factors [l I], notably eIF-2 [12-141. However, the question of whether the two processes, i.e.…”

Structure and function of peptide initiation factor 2: differential loss of activities during proteolysis and generation of a terminal fragment containing the phosphorylation sites of the alpha subunit.

“…One is a ribosome-bound enzyme that is activated by double-stranded RNA (5,6) and appears to be similar or identical to the enzyme that is induced by interferon in other cells (7)(8)(9)(10). The other protein kinase is activated in the absence of heme (2,5,11,12) and is associated with the heme-controlled repressor, HCR (13). Phosphorylation of eIF2a by either protein kinase blocks peptide initiation at a step that is dependent upon eIF-2.…”

“…This is a modification (21) of the procedure described by Laemmli (22). The details of the electrophoresis and autoradiography have been described (2,23).…”